High-level expression of recombinant human FK-binding protein from a fusion precursor |
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Authors: | Rohinton Edalji Tami J Pilot-Matias Steven D Pratt David A Egan Jean M Severin Earl G Gubbins Andrew M Petros Stephen W Fesik Neal S Burres and Thomas F Holzman |
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Institution: | (1) Protein Biochemistry, Structural Biology Unit, Abbott Laboratories, 60064 Abbott Park, Illinois;(2) Molecular Biology, Abbott Laboratories, 60064 Abbott Park, Illinois;(3) NMR Research, Drug Design and Delivery, Abbott Laboratories, 60064 Abbott Park, Illinois;(4) Anti-Infective Research, Pharmaceutical Products Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(5) Corporate Molecular Biology, Abbott Diagnostics Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(6) Protein Biochemistry, D-46Y, AP-10-1, Discovery Research, Abbott Laboratories, 60064 Abbott Park, Illinois |
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Abstract: | The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L inEscherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed inE. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to 3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind 3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein. |
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Keywords: | FK-binding protein CKS-FKBP fusion protein limited proteolysis peptidyl prolyl isomerase |
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